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Probing RNA Conformational Equilibria within the Functional Cellular Context.

Laura R Ganser1, Chia-Chieh Chu1, Hal P Bogerd2

  • 1Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.

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|February 27, 2020
PubMed
Summary
This summary is machine-generated.

Researchers developed a new method to measure RNA excited states (ESs) in cells. This study shows ESs in HIV-1 RNAs form in cells similarly to in vitro, suggesting targeted stabilization could be an anti-HIV therapy.

Keywords:
HIV-1 transactivation response elementRNA drug discoveryRNA dynamicsRNA structureRRERev response elementTARcellular activityconformational switchesexcited statestructure mappingtransactivation

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • RNA Biology

Background:

  • Low-abundance, short-lived non-native RNA conformations, termed excited states (ESs), are increasingly observed and implicated in RNA folding and biological functions.
  • Understanding the cellular abundance and role of these RNA excited states is crucial for comprehending RNA regulatory mechanisms.

Purpose of the Study:

  • To develop and validate an approach for assessing the relative abundance of RNA excited states (ESs) within the functional cellular context.
  • To investigate the in vivo relevance of RNA excited states for HIV-1 regulatory RNAs, specifically the transactivation response element (TAR) and the Rev response element (RRE).

Main Methods:

  • Utilized Nuclear Magnetic Resonance (NMR) spectroscopy to quantify the bias of substitution mutations toward inactive ESs in vitro.
  • Employed cellular activity assays of ES-stabilizing mutants as an indirect measure of cellular conformational equilibria.
  • Incorporated compensatory mutations to control for sequence-dependent effects and isolate conformational changes.

Main Results:

  • Demonstrated that RNA excited states (ESs) of HIV-1 TAR and RRE likely form in cells at abundances comparable to in vitro measurements.
  • Showed that targeted stabilization of these ESs could potentially serve as a therapeutic strategy against HIV-1.
  • Validated a novel approach for studying RNA conformational dynamics in a cellular environment.

Conclusions:

  • RNA excited states (ESs) are present and functionally relevant in the cellular context for HIV-1 regulatory RNAs.
  • The developed methodology provides a powerful tool for assessing RNA conformational equilibria in vivo.
  • Targeted stabilization of RNA excited states presents a promising avenue for the development of novel anti-HIV therapeutics.